Thelpers 1 (Th1) cells play a pro-atherogenic role, whereas regulatory T cells (Treg) demonstrate anti-inflammatory effects. The role of Th17 (CD4+IL-17+) cells in atherosclerosis remains controversial. Evidence suggests an existence of the cytokine-dependent Treg/Th17 cell plasticity. To date, the effects of atherosclerosis on the Treg/Th17 balance are unclear. The role of Th17 cells in the Th1- prevalent response in atherogenesis is not well defined. Our recent data reveals an increase of Th17 cells in apolipoprotein E (Apoe-/-) mice and a pro-atherogenic role of IL-17A. Our data implicates a function for plasma-derived cytokines in the regulation of the Treg/Th17 balance. The presence of IFN3+IL-17A+ T cells in Apoe-/- mice indicates that Th17 cells retain plasticity and may cooperate with Th1 cells. We hypothesize that atherosclerotic conditions shift the reciprocal Th17/Treg cell balance towards Th17 cells. These Th17 cells promote Th1 cell homing into aortas and together with Th1 cells provide a cooperative immune response during atherogenesis. Aim 1: To what extent do atherosclerotic conditions shift the balance of Treg/Th17 cells towards generation of Th17 cells? To examine the Treg/Th17 reciprocal regulation in atherogenesis, naive T cells (or natural or inducible Treg) from Foxp-3YFP-CreR26Y+/+ and Foxp-3YFP-CreR26Y-/- mice will be adoptively transferred into either C57BL/6 or Apoe-/- mice; allowing us to track cells that were previously Treg or currently express Foxp-3. The number of generated donor Treg, Th17 cells or cells converted from Treg to Th17 cells will be assessed. We will investigate the role of plasma cytokines in the regulation of Treg/Th17 balance and examine the interactions between transcription factors that regulate this reciprocal balance. We will show that shifting of the Treg/Th17 cell balance towards Treg via all-trans retinoic acid treatment will reduce the aortic inflammatory responses. Aim 2: To demonstrate a synergistic response of Th17 and Th1 cells in atherogenesis. We will examine IL-17-dependent homing of Th1 cells into aortas using adoptive transfer of Th1 cells into Il17a-/-Apoe-/- and Apoe-/- mice. We will analyze the immune response and Th1 chemokine expression in the aortas of Il17a-/-Apoe-/- and Apoe-/- mice. Next, we will adoptively transfer Th1 or Th17 or Th1+Th17 cells into Apoe-/- mice to examine a role of synergistic Th1/Th17 responses in the aortic inflammation. We will identify the levels of reprogramming of Th17 to IL-17+IFN3+ or Th1 cells by adoptive transfer of Th17 cells into Apoe-/- mice and examine IFN3+IL-17+ inflammatory cell profile. The proposed research is innovative, since the interaction of Th17 with Th1 and Treg cells in atherosclerosis has not been described. This proposal utilizes unique Foxp-3YFP-CreR26Y mice and new IL-17A-deficient Apoe-/- mice generated in our lab. Our project will provide new mechanistic data about IL-17A involvement in atherogenesis and highlight the unexpected role of Th17 cells in the Th1/Treg responses in this disease. PUBLIC HEALTH RELEVANCE: Atherosclerosis is an inflammatory chronic disease of the wall of large- and medium-sized arteries. This study will focus on the regulation of the reciprocal balance between suppressor T regulatory cells and pro- inflammatory Th17 cells, and synergistic effects of Th1 and Th17 cells in the development and progression of atherosclerosis. Anticipated results will help to understand the role of different T cell subsets and their reciprocal regulation in the immune response during atherosclerosis and will facilitate new approaches towards the prevention and treatment of the disease.